Translocation of macromolecules between cells is a major area of biomedical interest. The focus of study in this laboratory is a transfer system located at the Agrobacterium tumefaciens envelope which exports oncogenic DNA as a nucleoprotein particle, the T-complex, during infection of plant cells. The T-complex transporter is an especially attractive model system for detailed mechanistic studies of macromolecular transport. The T-complex transporter is a member of a newly-recognized family of medically-important transporters. Collectively, these transporters are responsible for conjugal transmission of broad-host-range plasmids between Gram-negative bacteria, and the export of at least one multisubunit toxin, the pertussis toxin of Bordetella pertussis. The T-complex transporter itself displays a novel versatility both with respect to substrate selection and target cell recognition. In addition to T-complexes, IncQ plasmid transfer intermediates and at least two proteins are recognized as translocation-competent substrates. Substrates are delivered to a wide range of plant species, other bacteria, and fission and budding yeast. Transport is a contact-dependent process that appears to require an extracellular pilus for mediating donor-recipient cell contacts. The overall goal of work in the PI's laboratory is to understand how this transporter is assembled, how it recognizes substrates, and how it directs the movement of substrates across the Gram-negative envelope to recipient cells. Dr. Christie has shown that ten of eleven products of the virB operon are essential for substrate translocation, and has supplied genetic and biochemical evidence that these proteins assemble as a large multimeric complex at the cell envelope. Toward definition of the transporter architecture, he will use a combination of molecular genetic and biochemical approaches to identify specific contacts among the VirB proteins. He will begin by identifying subunit interactions of two cytoplasmic membrane bound ATPases, VirB4 and VirB11, with other transporter components. He will use a large collection of mutations in these ATPases to analyze the contributions of ATP hydrolysis to transport with both in vitro and in vivo approaches. The PI will attempt to gain direct biochemical evidence for interactions between one of the translocation-competent substrates and components of the transport system. Finally, he plans to test and refine steps of a proposed biogenesis pathway for the T-complex transport system that was developed from two key discoveries in his laboratory, first, that the outer membrane lipoprotein, VirB7, forms an intermolecular disulfide bridge with VirB9, and second, that the VirB7/VirB9 heterodimer is critical for assembly of a stabilized transport complex.